Measuring instrument and fluoremetric method

ABSTRACT

A measuring instrument that comprises a light source unit  1  capable of emitting light having different wavelengths, a light receiving unit  2  that outputs an electrical signal corresponding to an intensity of transmitted light or radiated light from a sample  6  mixed with a plurality of coloring matters, and a calculation section  3 , is used. The calculation section  3  uses a previously calculated correction coefficient to calculate the fluorescence intensity of transmitted light or radiated light for each coloring matter. The correction coefficient is calculated based on an electrical signal output by the light receiving unit  2  when a plurality of correction samples are irradiated with light having different wavelengths, each correction sample being mixed with any one of the coloring matters and the respective mixed coloring matters being different from one another.

TECHNICAL FIELD

The present invention relates to a measuring instrument for measuring anintensity of transmitted light or radiated light for each coloringmatter when a sample mixed with a plurality of coloring matters isirradiated with light having wavelengths corresponding to the respectivecoloring matters. More particularly, the present invention relates to afluorescence measuring instrument and a fluorometric method ofirradiating a sample mixed with a plurality of fluorescent coloringmatters, with light having excitation wavelengths of the fluorescentcoloring matters, to measure fluorescence excited by the light.

BACKGROUND ART

In recent years, analysis of various components, genetic diagnosis andthe like are performed by measurement of fluorescence, absorbance orreflectance. For example, in component analysis using fluorescencemeasurement, a sample mixed with a coloring matter (fluorescent coloringmatter) is irradiated with light, and an intensity of fluorescenceexcited by the light is measured to detect a material labeled with thecoloring matter (fluorescent coloring matter).

In component analysis using absorbance measurement, a sample mixed witha coloring matter is irradiated with light having a wavelengthcorresponding to the coloring matter, and an intensity of transmittedlight is measured to calculate absorbance, thereby detecting a materiallabeled with the coloring matter, as disclosed in, for example, JP No.9-21749A. In component analysis using reflectance measurement, anintensity of scattered light, instead of transmitted light, is measuredto calculate reflectance, thereby detecting a material labeled with thecoloring matter.

In the case of detection of a plurality of materials using theabove-described component analysis, a sample is mixed with a pluralityof different coloring matters that vary depending on the materials to bedetected, and the sample is irradiated with light corresponding to eachcoloring matter separately.

In the case of fluorescence measurement, component analysis is performedby irradiating a sample mixed with a plurality of coloring matters(fluorescent coloring matters) that have different excitationwavelengths and fluorescence wavelengths, with light having theexcitation wavelength of each coloring matter separately, and measuringa fluorescence intensity of the coloring matter, as disclosed in, forexample, JP 2000-503774A.

In the case of absorbance measurement, component analysis is performedby irradiating a sample mixed with a plurality of coloring mattershaving different absorption wavelengths, with light having theabsorption wavelength of each coloring matter separately, to measure anintensity of transmitted light for each coloring matter.

However, in general, the excitation wavelength, absorption wavelengthand reflection wavelength of a coloring matter have a certain width.Therefore, in fluorescence measurement, if the coloring matters(fluorescent coloring matters) used have close excitation peakwavelengths, when a certain coloring matter is excited by light with itsexcitation wavelength, other coloring matter(s) also may be excited bythe light. In this case, the resultant fluorescence intensity is a valueobtained by combining the fluorescence intensity of each excitedcoloring matter, thereby making it difficult to perform accuratecomponent analysis, genetic diagnosis or the like.

The same is true of absorbance measurement and reflectance measurement.Specifically, the resultant intensity of transmitted light or scatteredlight is a value obtained by combining the intensity of transmittedlight or scattered light for each coloring matter, thereby making itdifficult to perform accurate component analysis, genetic diagnosis orthe like.

An object of the present invention is to provide a measuring instrumentand a fluorometric method capable of separating and measuring an actualintensity of each coloring matter from a combined value of the intensityof transmitted light or radiated light.

DISCLOSURE OF INVENTION

To achieve the above-described object, a measuring instrument of thepresent invention is a measuring instrument for measuring an intensityof transmitted light or radiated light for each coloring matter when asample mixed with a plurality of coloring matters is irradiated withlight having different wavelengths. The measuring instrument comprises alight source unit capable of irradiating the sample with the lighthaving the different wavelengths; a light receiving unit that receivesthe transmitted light or the radiated light and outputs an electricalsignal corresponding to the intensity of the received light; and acalculation section. The calculation section calculates the intensity ofthe transmitted light or the radiated light for each of the coloringmatters using a correction coefficient that is calculated based on anelectrical signal output by the light receiving unit when the lightsource unit irradiates each of a plurality of correction samples withlight having a different wavelength from each other, each correctionsample being mixed with one of the plurality of coloring matters and themixed coloring matters being different from one another.

In the measuring instrument of the present invention, the sample may bemixed with a plurality of fluorescent coloring matters having differentexcitation wavelengths as the coloring matters. The light receiving unitmay receive fluorescence of the fluorescent coloring matters, and outputan electrical signal corresponding to a fluorescence intensity of thereceived fluorescence. The calculation section may calculate thefluorescence intensity of the fluorescence of each of the fluorescentcoloring matters emitted from the sample using a correction coefficientthat is calculated based on an electrical signal output by the lightreceiving unit when the light source unit irradiates each of a pluralityof correction samples, each correction sample being mixed with one ofthe plurality of fluorescent coloring matters and the respective mixedfluorescent coloring matters being different from one another, withlight having a corresponding excitation wavelength of the plurality offluorescent coloring matters. In this embodiment, the measuringinstrument of the present invention functions as a fluorometric device.

In the embodiment in which the measuring instrument functions as afluorescence measuring instrument, preferably, the correctioncoefficient is a matrix (a_(ij) (i=1, 2, . . . , n; j=1, 2, . . . , n))satisfying Expression (1) $\begin{matrix}{{\begin{bmatrix}a_{11} & a_{12} & a_{13} & a_{14} & \ldots & a_{1n} \\a_{21} & a_{22} & a_{23} & a_{24} & \ldots & a_{2n} \\a_{31} & a_{32} & a_{33} & a_{34} & \ldots & a_{3n} \\a_{41} & a_{42} & a_{43} & a_{44} & \ldots & a_{4n} \\\vdots & \vdots & \vdots & \vdots & \vdots & \vdots \\a_{n1} & a_{n2} & a_{n3} & a_{n4} & \ldots & a_{nn}\end{bmatrix}\begin{bmatrix}Y_{1} \\Y_{2} \\Y_{3} \\Y_{4} \\\vdots \\Y_{n}\end{bmatrix}} = \begin{bmatrix}X_{1} \\X_{2} \\X_{3} \\X_{4} \\\vdots \\X_{n}\end{bmatrix}} & (1)\end{matrix}$where the plurality of fluorescent coloring matters mixed in the sampleare given numbers 1 to n, and when the light source unit irradiates thesample with light having an excitation wavelength of a k-th fluorescentcoloring matter (k=1, 2, . . . , n), an output value of the electricalsignal output by the light receiving unit is represented by X_(k), and afluorescence intensity of the k-th fluorescent coloring matter isrepresented by Y_(k). The calculation section substitutes the matrix(a_(ij)) and the output values X₁ to X_(n) into Expression (1) tocalculate the fluorescence intensities Y₁ to Y_(n) as the fluorescenceintensities of the fluorescent coloring matters.

Further, in the embodiment in which the measuring instrument functionsas a fluorescence measuring instrument, preferably, the measuringinstrument has a light amount monitor that detects a light amount oflight emitted by the light source unit and outputs a signal to thecalculation section. The calculation section corrects the output valuesX₁ to X_(n) or the matrix elements a₁₁ to a_(nn) based on the signaloutput by the light amount monitor.

Next, to achieve the above-described object, a fluorometric method ofthe present invention is a method for measuring a fluorescence intensityof fluorescence of each of a plurality of fluorescent coloring matters,the fluorescence being emitted from a sample mixed with the plurality offluorescent coloring matters having different excitation wavelengths, byusing a light source unit capable of emitting light having differentwavelengths and a light receiving unit that receives the fluorescence ofthe fluorescent coloring matters and outputs an electrical signalcorresponding to the fluorescence intensity of the receivedfluorescence. The method comprises calculating the fluorescenceintensity of the fluorescence of each fluorescent coloring matteremitted from the sample using a correction coefficient. The correctioncoefficient is calculated based on an electrical signal output by thelight receiving unit when the light source unit irradiates each of aplurality of correction samples, each correction sample being mixed withone of the plurality of fluorescent coloring matters and the respectivemixed fluorescent coloring matters being different from one another,with light having a corresponding excitation wavelength of the pluralityof fluorescent coloring matters.

In the fluorometric method of the present invention, preferably, thecorrection coefficient is a matrix (a_(ij) (i=1, 2, . . . , n; j=1, 2, .. . , n)) satisfying Expression (1) where the plurality of fluorescentcoloring matters mixed in the sample are given numbers 1 to n, and whenthe light source unit irradiates the sample with light having anexcitation wavelength of a k-th fluorescent coloring matter (k=1, 2, . .. , n), an output value of the electrical signal output by the lightreceiving unit is represented by X_(k), and a fluorescence intensity ofthe k-th fluorescent coloring matter is represented by Y_(k), and thematrix (a_(ij)) and the output values X₁ to X_(n) are substituted intoExpression (1) to calculate the fluorescence intensities Y₁ to Y_(n) asthe fluorescence intensities of the fluorescent coloring matters.

Further, in the fluorometric method of the present invention,preferably, the output values X₁ to X_(n) or the matrix elements a₁₁ toa_(nn) are corrected based on a light amount of light emitted by thelight source unit.

Furthermore, the present invention may be a program that implements thefluorometric method of the present invention. By installing the programinto a computer and executing the program, the fluorometric method ofthe present invention can be performed. Note that the term “coloringmatter” as used herein includes a fluorescent coloring matter for use influorescence measurement in addition to coloring matters for use inabsorbance measurement and reflectance measurement. When only afluorescent coloring matter is specified among the “coloring matters”,the term “fluorescent coloring matter” is used.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a structural diagram showing a fluorescence measuringinstrument according to an embodiment of the measuring instrument of thepresent invention.

FIG. 2 is a flowchart showing a fluorescence measurement processperformed by a fluorescence measuring instrument of FIG. 1.

FIG. 3 is a flowchart showing a correction coefficient calculationprocess performed in the fluorescence measuring instrument of FIG. 1.

FIG. 4 is a flowchart showing a light amount correction valuecalculation process performed in the fluorescence measuring instrumentof FIG. 1.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, an exemplary measuring instrument and fluorometric methodof the present invention will be described with reference to theaccompanying drawings. Note that, in the following description, themeasuring instrument of the present invention is a fluorescencemeasuring instrument, for example. Firstly, a structure of afluorescence measuring instrument according to an embodiment of themeasuring instrument of the present invention will be described withreference to FIG. 1. FIG. 1 is a structural diagram showing thefluorescence measuring instrument according to an embodiment of themeasuring instrument of the present invention.

The fluorescence measuring instrument of FIG. 1 is an instrument formeasuring a fluorescence intensity of fluorescence emitted from eachfluorescent coloring matter in a sample 6. As shown in FIG. 1, thefluorescence measuring instrument comprises a light source unit 1, alight receiving unit 2, a calculation section 3, a display section 4, areaction vessel 5, and a light amount monitor 7.

The sample 6 mixed with a plurality of fluorescent coloring matters isplaced in the reaction vessel 5. In the example of FIG. 1, the sample ismixed with four fluorescent coloring matters shown in Table 1 below.Note that, in the present invention, the fluorescent coloring mattersmixed into a sample are not limited to those shown in Table 1 below andthe number of the fluorescent coloring matters also is not limited. Inthe present invention, a required number of appropriate fluorescentcoloring matters can be selected, depending on the purpose thefluorescence measurement or the like. TABLE 1 Name of fluorescentcoloring matter FAM JOE TAMRA ROX Excitation peak 470 500 530 560wavelength [nm] Fluorescence 520 550 580 610 peak wavelength [nm]

The light source unit 1 has a structure capable of emitting light havingdifferent wavelengths and can irradiate a sample with light havingexcitation wavelengths of fluorescent coloring matters mixed in thesample. In the example of FIG. 1, the light source unit 1 compriseslight emitting devices 11 a to 11 d, dichroic mirrors 12 a to 12 d, anda total reflection mirror 13.

The light emitting devices 11 a to 11 d emit light in response to aninstruction of the calculation section 3. The light is used to excitethe fluorescent coloring matters mixed in the sample 6. The lightemitting devices 11 a to 11 d are disposed in a manner that causes lightbeams emitted by the light emitting devices to be directed in parallelto one another. The light emitting devices 11 a to 11 d emit lighthaving wavelengths different from one another. The individualwavelengths are set to be the excitation wavelength of one of thefluorescent coloring matters mixed in the sample. Specifically, thelight emitting device 11 a emits light having the excitation wavelengthof FAM, the light emitting device 11 b emits light having the excitationwavelength of JOE, the light emitting device 11 c emits light having theexcitation wavelength of TAMRA, and the light emitting device 11 d emitslight having the excitation wavelength of ROX.

The dichroic mirrors 12 a to 12 d have (high-pass) characteristics thatallow only light having a specific wavelength or less to be reflected.The maximum wavelength of light that can be reflected by the dichroicmirror is increased in the order: 12 a, 12 b, 12 c, 12 d.

Therefore, light emitted from each of the light emitting devices 11 a to11 d travels on the same optical path to enter the total reflectionmirror 13, and is reflected from the total reflection mirror 13 to enterthe reaction vessel 5. An amount of light emitted from the lightemitting devices 11 a to 11 d is monitored by the light amount monitor7. The light amount monitor 7 detects the amount of light emitted by thelight emitting devices 11 a to 11 d and outputs a signal to thecalculation section 3.

Note that, in the example of FIG. 1, since the number of fluorescentcoloring matters used is four, the number of light emitting devicesconstituting the light source unit 1 is also four. The number ofdichroic mirrors is also four, corresponding to the number of lightemitting devices. Note that, in the present invention, the number oflight emitting devices and the number of dichroic mirrors are notlimited to this, and are determined depending on the number offluorescent coloring matters used. As the light emitting devices 11 a to11 d, light emitting diodes or semiconductor lasers are used preferably,and xenon lamps or halogen lamps also can be used.

The light receiving unit 2 receives fluorescence emitted from thereaction vessel 5, and outputs an electrical signal corresponding to afluorescence intensity of the received fluorescence. In the example ofFIG. 1, the light receiving unit comprises light receiving devices 14 ato 14 d, dichroic mirrors 15 a to 15 d, and a total reflection mirror16.

In the example of FIG. 1, the dichroic mirrors 15 a to 15 d have(low-pass) characteristics that allow light having a specific wavelengthor more to be reflected. The minimum wavelength of light that can bereflected by the dichroic mirror is increased in the order: 15 d, 15 c,15 b, 15 a. The light receiving devices 14 a to 14 d are photodiodes andare arranged in a manner such that light reflected from one dichroicmirror enters the light receiving surface (not shown) of one lightreceiving device.

Therefore, fluorescence emitted from the reaction vessel 5 is reflectedfrom the total reflection mirror 16, and thereafter is reflected fromone of the dichroic mirrors 15 a to 15 d, depending on the wavelength ofthe fluorescence to enter a corresponding light receiving device. As aresult, each light receiving device outputs an electrical signalcorresponding to a fluorescence intensity of fluorescence to thecalculation section 3.

The calculation section 3 calculates fluorescence intensity based on theelectrical signal output from the light receiving unit 2. A result ofthe calculation is displayed on the display section 4. The displaysection 4 is a liquid crystal display apparatus, a CRT or the like.

Next, a fluorometric method of the present invention will be describedwith reference to FIGS. 2 to 4. Note that the fluorometric method of thepresent invention can be performed by operating the fluorescencemeasuring instrument of FIG. 1. Therefore, in the following description,an operation of the fluorescence measuring instrument of FIG. 1 will bedescribed.

FIG. 2 is a flowchart showing a fluorescence measurement processperformed by the fluorescence measuring instrument of FIG. 1. As shownin FIG. 2, the calculation section 3 of the fluorescence measuringinstrument initially determines whether or not a correction coefficienthas been calculated (step S1). The correction coefficient is used forcalculation of fluorescence intensity for each fluorescent coloringmatter based on an electrical signal output by the light receiving unit2 when a sample mixed with a plurality of fluorescent coloring mattersis irradiated with light having an excitation wavelength of eachfluorescent coloring matter.

As used herein, the term “fluorescence intensity of each fluorescentcoloring matter” refers to, not a combined value obtained inconventional fluorescence measurement, but an intensity of fluorescenceemitted only by a fluorescent coloring matter whose excitationwavelength corresponds to the wavelength of the light when a sample isirradiated with light. In the present invention, the above-describedcorrection coefficient is used to separate an actual fluorescenceintensity from a combined value as described below.

In the example of FIG. 2, the correction coefficient is a matrix(a_(ij)) that satisfies the above-described expression (1). Note that,in the example, the number of fluorescent coloring matters mixed in asample is four as described above. Therefore, the four fluorescentcoloring matters mixed in a sample are given numbers 1 to 4 in order ofexcitation wavelength from the shortest. The correction coefficient is amatrix (a_(ij) (i=1, 2, 3, 4; j=1, 2, 3, 4)) that satisfies Expression(2) below: $\begin{matrix}{{\begin{bmatrix}a_{11} & a_{12} & a_{13} & a_{14} \\a_{21} & a_{22} & a_{23} & a_{24} \\a_{31} & a_{32} & a_{33} & a_{34} \\a_{41} & a_{42} & a_{43} & a_{44}\end{bmatrix}\begin{bmatrix}Y_{1} \\Y_{2} \\Y_{3} \\Y_{4}\end{bmatrix}} = \begin{bmatrix}X_{1} \\X_{2} \\X_{3} \\X_{4}\end{bmatrix}} & (2)\end{matrix}$where, when the light source unit 1 irradiates a sample with lighthaving the excitation wavelength of a k-th fluorescent coloring matter(k=1, 2, 3, 4), an output value of an electrical signal output by thelight receiving unit 2 is represented by X_(k), and the fluorescenceintensity of the k-th fluorescent coloring matter is represented byY_(k).

When it is determined in step S1 that the correction coefficient has notyet been calculated, the calculation section 3 performs a correctioncoefficient calculation process (step S2), and thereafter, step S3below. Note that the correction coefficient calculation process of stepS2 specifically will be described below.

On the other hand, when it is determined that the correction coefficienthas been calculated in step S1, the calculation section 3 causes each ofthe light emitting devices 11 a to 11 d to emit light and causes thelight amount monitor 7 to measure the amount of light. When the measuredlight amount is varied, the calculation section 3 calculates a lightamount correction value for correcting output values X₁ to X₄ describedbelow (step S3). Note that details of step S3 will be described below.

Next, the calculation section 3 causes each light emitting device of thelight source unit 1 to emit light having an excitation wavelength towarda sample in order to measure fluorescence intensity (step S4).Thereafter, the calculation section 3 receives an electrical signaloutput by the light receiving unit 2 to obtain its output value X_(k)(k=1 to 4) (step S5).

Note that, in the example of FIGS. 1 to 4, the output value of theelectrical signal output by the light receiving unit 2 is a digitalvalue that is obtained by I/V converting a current value of anelectrical signal output by the light receiving devices 14 a to 14 d andfurther A/D converting the resultant voltage value. However, the presentinvention is not limited to this. The output value of the electricalsignal output by the light receiving unit 2 may be a digital valueobtained by A/D converting the current value of the electrical signaloutput by the light receiving devices 14 a to 14 d.

Thereafter, the calculation section 3 determines whether or not all theoutput values X₁ to X₄ have been obtained (step S6). When not all theoutput values X₁ to X₄ have been obtained, the calculation section 3performs steps S4 and S5 again.

On the other hand, when all the output values X₁ to X₄ have beenobtained, the calculation section 3 substitutes the correctioncoefficient and the output values X₁ to X₄ obtained in step S5 into theabove-described Expression (2) to calculate fluorescence intensities Y₁to Y₄ of the fluorescent coloring matters (step S7). Note that, when alight amount correction value has been calculated in step S3, the outputvalues X₁ to X₄ or the matrix elements of the matrix (a_(ij) (i=1, 2, 3,4; j=1, 2, 3, 4)) corrected using the light amount correction value aresubstituted into Expression (2).

The above-described fluorescence measurement process is then ended, andthe fluorescence intensity of each fluorescent coloring matter isdisplayed on the display section 4. Thus, the fluorescence measuringinstrument and the fluorometric method of the present invention can beused to separate actual fluorescence intensity from a combined value,resulting in more accurate fluorescence measurement than conventionaltechniques.

Next, the correction coefficient calculation process in step S2 of FIG.2 will be described with reference to FIG. 3. FIG. 3 is a flowchartshowing the correction coefficient calculation process performed in thefluorescence measuring instrument of FIG. 1.

The correction coefficient calculation process is performed using aplurality of samples for correction. Each correction sample is mixedwith only one of the fluorescent coloring matters to be mixed in asample. The fluorescent coloring matters mixed in the respectivecorrection samples are different from one another. Specifically, in theexample of FIG. 3, a sample is mixed with four fluorescent coloringmatters as shown in Table 1, and therefore, four correction samples arerequired.

The correction coefficient calculation process is performed usingExpressions (3) to (6) below, which are obtained by expanding Expression(2).a ₁₁ Y ₁ +a ₁₂ Y ₂ +a ₁₃ Y ₃ +a ₁₄ Y ₄ =X ₁  (3)a ₂₁ Y ₁ +a ₂₂ Y ₂ +a ₂₃ Y ₃ +a ₂₄ Y ₄ =X ₂  (4)a ₃₁ Y ₁ +a ₃₂ Y ₂ +a ₃₃ Y ₃ +a ₃₄ Y ₄ =X ₃  (5)a ₄₁ Y ₁ +a ₄₂ Y ₂ +a ₄₃ Y ₃ +a ₄₄ Y ₄ =X ₄  (6)

As shown in FIG. 3, the calculation section 3 initially irradiates thecorrection sample with light from each light emitting device (step S11),and then obtains an output value of an electrical signal output by thelight receiving unit 2 (step S12). Thereafter, the calculation section 3substitutes the obtained output values into Expressions (3) to (6) (stepS13).

Next, the calculation section 3 determines whether or not output valueshave been obtained from all the correction samples (step S14). Whenoutput values have been obtained from not all the correction samples,steps S11 to S13 are performed again. When output values have beenobtained from all the correction samples, step S15 is performed.

Steps S11 to S13 will be described specifically. The calculation section3 initially irradiates a correction sample mixed only with a firstfluorescent coloring matter (FAM), with light having the excitationwavelengths of first to fourth fluorescent coloring matters using thelight source unit 1. In this case, output values of output electricalsignals are represented by F1 to F4, corresponding to the names of thefluorescent coloring matters. The calculation section 3 substitutes theoutput values F1 to F4 into X₁ to X₄ of Expressions (3) to (6). Also inthis case, since the correction sample is mixed only with the firstfluorescent coloring matter, Y₂=Y₃=Y₄=0 (zero) in Expressions (3) to(6). Therefore, Expressions (7) to (10) below are obtained.a ₁₁ Y ₁ =F1  (7)a ₂₁ Y ₁ =F2  (8)a ₃₁ Y ₁ =F3  (9)a ₄₁ Y ₁ =F4  (10)

Similarly, the calculation section 3 irradiates a correction samplemixed only with a second fluorescent coloring matter (JOE), a correctionsample mixed with a third fluorescent coloring matter (TAMRA), and acorrection sample mixed only with a fourth fluorescent coloring matter(ROX), with light having the excitation wavelengths of the first tofourth fluorescent coloring matters using the light source unit 1,obtains output values of output electrical signals, and substitutes theobtained output values into Expressions (3) to (6). The output values inthese cases are represented by J1 to J4, T1 to T4, and R1 to R4,respectively. In this case, Expressions (11) to (22) below are obtained.a ₁₂ Y ₂ =J1  (11)a ₂₂ Y ₂ =J2  (12)a ₃₂ Y ₂ =J3  (13)a ₄₂ Y ₂ =J4  (14)a ₁₃ Y ₃ =T1  (15)a ₂₃ Y ₃ =T2  (16)a ₃₃ Y ₃ =T3  (17)a ₄₃ Y ₃ =T4  (18)a ₁₄ Y ₄ =R1  (19)a ₂₄ Y ₄ =R2  (20)a ₃₄ Y ₄ =R3  (21)a ₄₄ Y ₄ =R4  (22)

Next, in step S15, the calculation section 3 uses Expressions (7) to(22) obtained in step S13 to calculate a matrix (a_(ij) (i=1, 2, 3, 4;j=1, 2, 3, 4)) that satisfies Expression (2), as a correctioncoefficient.

Specifically, the calculation section 3 sets a₁₁=a₂₂=a₃₃=a₄₄=1 tocalculate the correction coefficient. For example, a ratio ofa₁₁:a₁₂:a₁₃:a₁₄ is determined, depending on a fluorescent coloringmatter used. Therefore, the calculation section 3 calculates a₁₂=J1/F1,a₁₃=T1/F1, and a₁₄=R1/F1.

Similarly, the calculation section 3 calculates a₂₁=F2/J2, a₂₃=T2/J2,and a₂₄=R2/J2. The calculation section 3 also calculates a₃₁=F3/T3,a₃₂=J3/T3, and a₃₄=R3/T3. Further, the calculation section calculatesa₄₁=F4/R4, a₄₂=J4/R4, and a₄₃=T4/R4.

The above-described correction coefficient calculation process is thenended. Note that the correction coefficient calculation process can beperformed before shipment of the fluorescence measuring instrument ofthe present invention. In this case, it is preferable that thecorrection coefficient previously is stored in a memory in thefluorescence measuring instrument before product shipment. Also in thiscase, steps S1 and S2 do not have to be performed in the fluorescencemeasurement process of FIG. 2.

Next, correction of an output value based on a light amount, which isshown in step S3 of FIG. 2, will be described with reference to FIG. 4.FIG. 4 is a flowchart showing a light amount correction valuecalculation process performed in the fluorescence measuring instrumentof FIG. 1.

When the light amounts of the light emitting devices 11 a to 11 d varywith time and environment, the ratio of a₁₁:a₂₁:a₃₁:a₄₁, the ratio ofa₁₂:a₂₂:a₃₂:a₄₂, the ratio of a₁₃:a₂₃:a₃₃:a₄₃, and the ratio ofa₁₄:a₂₄:a₃₄:a₄₄ are influenced, so that it is no longer possible tocalculate a fluorescence brightness accurately. To prevent this, thelight amount correction value calculation process of FIG. 4 isperformed.

Specifically, as shown in FIG. 4, the calculation section 3 initiallymeasures a current light amount ratio of the light emitting devices 11a, 11 b, 11 c and 11 d based on a signal from a light amount monitorwhere the light amount ratio of the light emitting devices when thecorrection coefficient was determined is assumed to be 1:1:1:1(reference value) (step S21).

Next, the calculation section 3 determines whether or not the obtainedlight amount ratio is changed from 1:1:1:1 (step S22). When the lightamount ratio is not changed, the calculation section 3 ends the process.On the other hand, when the light amount ratio is changed, thecalculation section 3 calculates a light amount correction valuecorresponding to a change width (step S23).

For example, it is assumed that the ratio of light amounts of the lightemitting devices 11 a, 11 b, 11 c and 11 d is 1:2:3:4. In this case, inorder to return the light amount ratio to a reference value, the lightamount of each light emitting device needs to be multiplied by 1/1, 1/2,1/3, or 1/4. Therefore, the light amount correction values are 1/1, 1/2,1/3, and 1/4. Note that, when the light receiving devices 14 a to 14 dhave a low level of sensitivity, a variation in light amount has a smallinfluence on fluorescence intensity. When the sensitivity is high, thereverse is true. Therefore, it can be said that the light amountcorrection value preferably is determined, taking into consideration thesensitivities of the light receiving devices 14 a to 14 d.

Therefore, the calculation section 3 substitutes (1/1)X₁, (1/2)X₂,(1/3)X₃ and (1/4)X₄ instead of X₁, X₂, X₃ and X₄ into Expression (2) tocalculates the fluorescence intensities Y₁ to Y₄ in step S7 of FIG. 2.Alternatively, the calculation section 3 substitutes (1/1)a_(i1),(1/2)a_(i2), (1/3)a_(i3), and (1/4)a_(i4) instead of matrix elementsa_(i1), a_(i2), a_(i3), and a_(i4) into the matrix (a_(ij)(i=1, 2, 3, 4;j=1, 2, 3, 4)) that satisfies Expression (2) to calculate thefluorescence intensity Y₁ to Y₄.

Thus, according to the fluorescence measuring instrument and thefluorometric method of FIG. 1, even if the light amounts of lightemitting devices vary, fluorescence intensity can be calculated based oncorrected light amounts of the light emitting devices, thereby making itpossible to improve the accuracy of fluorescence measurement further.

Note that the fluorescence measuring instrument of FIG. 1 can beimplemented by installing a program that causes a computer connected tothe light source unit 1 and the light receiving unit 2 to execute stepsS1 to S7 of FIG. 2 and performing the program. In this case, the CPU(central processing unit) of the computer functions as the calculationsection 3.

In the above-described embodiments, the fluorescence measuringinstrument and the fluorometric method are described as examples. Notethat the present invention is not limited to the examples. The presentinvention may be applied to a measuring instrument or method usingabsorbance or reflectance measurement. Specifically, according to thepresent invention, even when a sample is mixed with a plurality ofcoloring matters having different absorption wavelengths or reflectionwavelengths, a correction coefficient can be calculated in the samemanner as described above and the intensity of transmitted light orscattered light can be calculated for each coloring matter. Further, anabsorbance can be calculated based on the calculated intensity oftransmitted light, and a reflectance can be calculated based on theintensity of scattered light.

INDUSTRIAL APPLICABILITY

As described above, according to the measuring instrument and thefluorometric method of the present invention, even when a sample ismixed with a plurality of coloring matters (e.g., fluorescent coloringmatters), the actual intensity of each coloring matter can be separatedfrom the resultant (fluorescence) intensity of transmitted light orradiated light. Therefore, by using the measuring instrument and thefluorometric method of the present invention, it is possible to performmore accurate component analysis, genetic diagnosis and the like thanwith conventional techniques.

1. A measuring instrument for measuring an intensity of transmittedlight or radiated light for each coloring matter when a sample mixedwith a plurality of coloring matters is irradiated with light havingdifferent wavelengths, comprising: a light source unit capable ofirradiating the sample with the light having the different wavelengths;a light receiving unit that receives the transmitted light or theradiated light and outputs an electrical signal corresponding to theintensity of the received light; and a calculation section, wherein thecalculation section calculates the intensity of the transmitted light orthe radiated light for each of the coloring matters using a correctioncoefficient that is calculated based on an electrical signal output bythe light receiving unit when the light source unit irradiates each of aplurality of correction samples with light having a different wavelengthfrom one another, each correction sample being mixed with one of theplurality of coloring matters and the respective mixed coloring mattersbeing different from one another.
 2. The measuring instrument accordingto claim 1, wherein the sample is mixed with a plurality of fluorescentcoloring matters having different excitation wavelengths as the coloringmatters, the light receiving unit receives fluorescence of thefluorescent coloring matters, and outputs an electrical signalcorresponding to a fluorescence intensity of the received fluorescence,and the calculation section calculates the fluorescence intensity of thefluorescence of each of the fluorescent coloring matters emitted fromthe sample using a correction coefficient that is calculated based on anelectrical signal output by the light receiving unit when the lightsource unit irradiates each of a plurality of correction samples, eachcorrection sample being mixed with one of the plurality of fluorescentcoloring matters and the respective mixed fluorescent coloring mattersbeing different from one another, with light having a correspondingexcitation wavelength of the plurality of fluorescent coloring matters.3. The measuring instrument according to claim 2, wherein the correctioncoefficient is a matrix (a_(ij) (i=1, 2, . . . , n; j=1, 2, . . . , n))satisfying Expression (23): $\begin{matrix}{{\begin{bmatrix}a_{11} & a_{12} & a_{13} & a_{14} & \ldots & a_{1n} \\a_{21} & a_{22} & a_{23} & a_{24} & \ldots & a_{2n} \\a_{31} & a_{32} & a_{33} & a_{34} & \ldots & a_{3n} \\a_{41} & a_{42} & a_{43} & a_{44} & \ldots & a_{4n} \\\vdots & \vdots & \vdots & \vdots & \vdots & \vdots \\a_{n1} & a_{n2} & a_{n3} & a_{n4} & \ldots & a_{nn}\end{bmatrix}\begin{bmatrix}Y_{1} \\Y_{2} \\Y_{3} \\Y_{4} \\\vdots \\Y_{n}\end{bmatrix}} = \begin{bmatrix}X_{1} \\X_{2} \\X_{3} \\X_{4} \\\vdots \\X_{n}\end{bmatrix}} & (23)\end{matrix}$ where the plurality of fluorescent coloring matters mixedin the sample are given numbers 1 to n, and when the light source unitirradiates the sample with light having an excitation wavelength of ak-th fluorescent coloring matter (k=1, 2, . . . , n), an output value ofthe electrical signal output by the light receiving unit is representedby X_(k), and a fluorescence intensity of the k-th fluorescent coloringmatter is represented by Y_(k), and the calculation section substitutesthe matrix (aij) and the output values X₁ to X_(n) into Expression (23)to calculate the fluorescence intensities Y₁ to Y_(n) as thefluorescence intensities of the fluorescent coloring matters.
 4. Themeasuring instrument according to claim 3, having a light amount monitorthat detects a light amount of light emitted by the light source unitand outputs a signal to the calculation section, wherein the calculationsection corrects the output values X₁ to X_(n) or the matrix elementsa₁₁ to a_(nn) based on the signal output by the light amount monitor. 5.A fluorometric method for measuring a fluorescence intensity offluorescence of each of a plurality of fluorescent coloring matters, thefluorescence being emitted from a sample mixed with the plurality offluorescent coloring matters having different excitation wavelengths, byusing a light source unit capable of emitting light having differentwavelengths and a light receiving unit that receives the fluorescence ofthe fluorescent coloring matters and outputs an electrical signalcorresponding to the fluorescence intensity of the receivedfluorescence, the method comprising: calculating the fluorescenceintensity of the fluorescence of each fluorescent coloring matteremitted from the sample using a correction coefficient, wherein thecorrection coefficient is calculated based on an electrical signaloutput by the light receiving unit when the light source unit irradiateseach of a plurality of correction samples, each correction sample beingmixed with one of the plurality of fluorescent coloring matters and therespective mixed fluorescent coloring matters being different from oneanother, with light having a corresponding excitation wavelength of theplurality of fluorescent coloring matters.
 6. The fluorometric methodaccording to claim 5, wherein the correction coefficient is a matrix(a_(ij) (i=1, 2, . . . , n; j=1, 2, . . . , n)) satisfying Expression(24): $\begin{matrix}{{\begin{bmatrix}a_{11} & a_{12} & a_{13} & a_{14} & \ldots & a_{1n} \\a_{21} & a_{22} & a_{23} & a_{24} & \ldots & a_{2n} \\a_{31} & a_{32} & a_{33} & a_{34} & \ldots & a_{3n} \\a_{41} & a_{42} & a_{43} & a_{44} & \ldots & a_{4n} \\\vdots & \vdots & \vdots & \vdots & \vdots & \vdots \\a_{n1} & a_{n2} & a_{n3} & a_{n4} & \ldots & a_{nn}\end{bmatrix}\begin{bmatrix}Y_{1} \\Y_{2} \\Y_{3} \\Y_{4} \\\vdots \\Y_{n}\end{bmatrix}} = \begin{bmatrix}X_{1} \\X_{2} \\X_{3} \\X_{4} \\\vdots \\X_{n}\end{bmatrix}} & (24)\end{matrix}$ where the plurality of fluorescent coloring matters mixedin the sample are given numbers 1 to n, and when the light source unitirradiates the sample with light having an excitation wavelength of ak-th fluorescent coloring matter (k=1, 2, . . . , n), an output value ofthe electrical signal output by the light receiving unit is representedby X_(k), and a fluorescence intensity of the k-th fluorescent coloringmatter is represented by Y_(k), and the matrix (a_(ij)) and the outputvalues X₁ to X_(n) are substituted into Expression (24) to calculate thefluorescence intensities Y₁ to Y_(n) as the fluorescence intensities ofthe fluorescent coloring matters.
 7. The fluorometric method accordingto claim 6, wherein the output values X₁ to X₁ or the matrix elementsa₁₁ to a_(nn) are corrected based on a light amount of light emitted bythe light source unit.
 8. A program for causing a computer to measure afluorescence intensity of fluorescence of each of a plurality offluorescent coloring matters, the fluorescence being emitted from asample mixed with the plurality of fluorescent coloring matters havingdifferent excitation wavelengths using a light source unit capable ofemitting light having different wavelengths and a light receiving unitthat receives the fluorescence of the fluorescent coloring matters andoutputs an electrical signal corresponding to the fluorescence intensityof the received fluorescence, the program comprising, the step ofcalculating the fluorescence intensity of the fluorescence of eachfluorescent coloring matter emitted from the sample using a correctioncoefficient, wherein the correction coefficient is calculated based onan electrical signal output by the light receiving unit when the lightsource unit irradiates each of a plurality of correction samples, eachcorrection sample being mixed with one of the plurality of fluorescentcoloring matters and the respective mixed fluorescent coloring mattersbeing different from one another, with light having a correspondingexcitation wavelength of the plurality of fluorescent coloring matters.9. The program according to claim 8, wherein the correction coefficientis a matrix (a_(ij) (i=1, 2, . . . , n; j=1, 2, . . . , n)) satisfyingExpression (25): $\begin{matrix}{{\begin{bmatrix}a_{11} & a_{12} & a_{13} & a_{14} & \ldots & a_{1n} \\a_{21} & a_{22} & a_{23} & a_{24} & \ldots & a_{2n} \\a_{31} & a_{32} & a_{33} & a_{34} & \ldots & a_{3n} \\a_{41} & a_{42} & a_{43} & a_{44} & \ldots & a_{4n} \\\vdots & \vdots & \vdots & \vdots & \vdots & \vdots \\a_{n1} & a_{n2} & a_{n3} & a_{n4} & \ldots & a_{nn}\end{bmatrix}\begin{bmatrix}Y_{1} \\Y_{2} \\Y_{3} \\Y_{4} \\\vdots \\Y_{n}\end{bmatrix}} = \begin{bmatrix}X_{1} \\X_{2} \\X_{3} \\X_{4} \\\vdots \\X_{n}\end{bmatrix}} & (25)\end{matrix}$ where the plurality of fluorescent coloring matters mixedin the sample are given numbers 1 to n, and when the light source unitirradiates the sample with light having an excitation wavelength of ak-th fluorescent coloring matter (k=1, 2, . . . , n), an output value ofthe electrical signal output by the light receiving unit is representedby X_(k), and a fluorescence intensity of the k-th fluorescent coloringmatter is represented by Y_(k), and the matrix (a_(ij)) and the outputvalues X₁ to X_(n) are substituted into Expression (25) to calculate thefluorescence intensities Y₁ to Y_(n) as the fluorescence intensities ofthe fluorescent coloring matters.
 10. The program according to claim 9,wherein the output values X₁ to X_(n) or the matrix elements a₁₁ toa_(nn) are corrected based on a light amount of light emitted by thelight source unit.